The thioredoxin system is ubiquitous, and plays an essential role in maintaining thiol/disulfide redox homeostasis in cells. The Mycobacterium tuberculosis (M. tb) thioredoxin system has recently been suggested as a target for anti-tuberculosis (TB) drug development, by disrupting its ability to protect M. tb from the oxidative attacks of macrophages. But, while thioredoxin systems from some bacteria (ex. E. coli) and human is well-characterized, the mycobacterial system is not. The M. tb system is comprised of three thioredoxins (TrxA, TrxB and TrxC) and one thioredoxin reductase (TrxR). Drug discovery efforts targeting this system are hindered by the lack of available structures for TrxA and TrxB and of their complexes with TrxR. Furthermore, the differential roles of the three M. tb thioredoxins are not known, with some publications suggesting that TrxA may even be non-functional. The objective of this project is to define different structural/functional properties o the three M. tb thioredoxins, and to determine if TrxA is truly cryptic. We will use these structures a a foundation for structure-based identification of inhibitors of the entire M. tb thioredoxin syste, or specific complexes. Expected outcomes and impact: This project will produce a structural characterization of the M. tb thioredoxin system, and inhibitors as chemical genetic probes of function and anti-TB drug leads. Our Aims Are to: 1. Determine solution structures for all M. tuberculosis thioredoxins. Determine 4 NMR structures: TrxA and TrxB in both redox states (thiol/disulfide), and compare to our TrxC structures. 2. Determine structural models of M. tuberculosis TrxR/TrxN (where N= A, B) complexes, and compare to TrxR/TrxC. (a) Establish if and how TrxR binds the two TrxN's (both redox states). (b) Determine structural changes in TrxN, induced by TrxR binding. (c) Determine dynamics changes in NADPH cofactor (bound to TrxR), induced by TrxN binding. (d) Construct structural models for the two TrxN/TrxR complexes based on NMR chemical shift perturbations, for various dead end complexes to mimic intermediates in the catalytic cycle. 3. Identify inhibitors of the M. tuberculosis TrxR/Trx system. (a) Using the 3-dimensional structural models for the M. tuberculosis TrxR/TrxN system (and comparable M. smegmatis structures), computationally dock compounds to identify candidate inhibitors, (b) test candidate inhibitors in both NMR binding (titration) and enzymatic inhibition assays, to determine affinity, and (c) test compounds that have Kd < 50 mM in MIC (minimum inhibitory concentration) assays initially with M. smegmatis (then M. tuberculosis). PUBLIC HEALTH RELEVANCE: This study will yield a comprehensive structural characterization of the thioredoxin enzyme system from Mycobacterium tuberculosis, the causative agent of tuberculosis. Since the Mycobacterium tuberculosis thioredoxin system protects it from the oxidative attacks of human immune cells, it is currently being pursued as a promising new drug target. The structural data from this project will therefore be used to guide structure-based identification of inhibitors, potentially providing new therapeutic agents for treating mycobacterial infections, as well as chemical genetic probes to increase understanding of this enzyme system.